Fitc or tritc conjugated secondary antibody

HeLa cells were plated on glass coverslips and then transfected with 0.5 μg of vectors or heat stress (42°C, 1h). After incubation, cells were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100 in PBS, and then incubated with a blocking solution (Dako, Glostrup, Denmark). After incubation overnight with primary antibody for NICD (1:200, Millipore, Billerica, MA, USA) or YTHDF2 (1:200, MBL, Woburn, MA, USA) in blocking solution, cells were washed and incubated with FITC or TRITC-conjugated secondary antibodies (1:200, Jackson Immuno Research Laboratories, West Grove, PA, USA) for 1 h at room temperature. After staining with DAPI (Life Technologies, Carlsbad, CA, USA), cells were observed under a confocal microscope (Leica TCS SPE, Buffalo Grove, IL, USA).

Paraffin-embedded sections were incubated at 60°C for 2 h and then deparaffinised with xylene and rehydrated in a series of solutions containing descending concentrations of ethanol. For antigen retrieval, sections were pretreated with boiling citric acid buffer (10 mM, pH 6.0) for 15 min. After blocking with 10% normal donkey serum for 1 h at room temperature, sections were then incubated with the following primary antibodies overnight at 4°C: anti-LC3 antibodies (1:1,000 dilution) (Proteintech, Chicago, IL), anti-P62 antibodies (1:1,000, Proteintech, Chicago, IL), and anti-Atg7 antibodies (1:1,000, Proteintech, Chicago, IL). Sections were then incubated with FITC- or TRITC-conjugated secondary antibodies (Jackson ImmunoResearch, 1:100). Images were captured using a microscope attached to a charge-coupled device (CCD) camera (Leica, Germany).

For all experiments involving RD cells, the cells were fixed with 4% paraformaldehyde (Electron Microscopy Sciences) in PBS for seven minutes at room temperature prior to permeabilization with 0.5% Triton X-100 (Sigma) in PBS. Cells were then incubated with 1% BSA in PBS block solution for 30 minutes at room temperature and then incubated overnight with J2 or K1 antibody at a concentration of 2 μg/ml at 4°C. Appropriate FITC- or TRITC-conjugated secondary antibodies (Jackson ImmunoResearch) and DAPI stain (Sigma) were then used prior to visualization. For the human myoblast experiment, conditions were essentially the same, except that cells were fixed with 2% paraformaldehyde and co-incubated overnight with J2 or K1 and anti-DUX4 (E5-5) antibody. Cells were imaged on a Zeiss AxioPhot or, if indicated, a Leica TCS SP5 II confocal microscope and channel merging was performed using ImageJ software.

AIMP1/p43 antibodies are describes elsewhere [17] (link). HA, FLAG, and β-tubulin antibodies were purchased from Sigma. grp78 and gp96 antibodies were purchased from Santa Cruz Biotechnology. GST antibody was from Amersham Bioscience and V5 antibody was from Invitrogen. HRP-conjugated mouse or rabbit secondary antibody and FITC- or TRITC-conjugated secondary antibody were purchased from Jackson ImmunoResearch Laboratories. TGF-β was from R&D Systems and MG132 was from Calbiochem.

Specimens were fixed for 24 hrs with 10% buffered formalin before embedding in paraffin. Serial sections of 5 μm thick were obtained for histologic analysis. Hematoxylin&eosin (HE) staining involved standard procedures.
For immunohistochemistry, sections were incubated with the primary antibodies for Cat S (1:200), CD68 (1:200), Ki-67 (1:200), CD31 (1:200), Mac-2 (1:200), then incubated with the Dako ChemMateTM EnVision System (Dako, Glostrup, Denmark) for 30 min. Staining was visualized with use of diaminobenzidine and counterstaining with hematoxylin. Negative controls were omission of the primary antibody, non-immune IgG or secondary antibody only; in all cases, negative controls showed insignificant staining. The expressions of Cat S, CD68, Ki-67, CD31, Mac-2 were calculated as proportion of positive area to total tissue area for all measurements of the section.
For double immunofluorescence, 7 μm frozen tissue sections were permeabilized and blocked with 0.1% Triton X-100, 0.2% bovine serum albumin, and 5% normal donkey serum in PBS, then incubated with the primary antibodies F4/80 (1:100), LC-3 (1:200) and Cat S (1:200) overnight at 4°C, then FITC or TRITC-conjugated secondary antibody (Jackson Immuno Research Laboratories, West Grove, PA, USA) at 4°C for 1 hr in the dark, and coverslipped with DAPI-containing mounting medium.